Project summary/abstract Arterial calcification has increasingly been associated with poor outcomes in a wide variety of patient populations including those with coronary, aortic, and peripheral artery disease, as well as patients with diabetes, and renal failure. We previously demonstrated that calcification in peripheral arteries predicts major amputation even after adjusting for demographics, cardiovascular risk factors, and the ankle brachial index. During arterial calcification, medial smooth muscle cells (SMCs) undergo osteogenic transformation with loss of phenotype-specific markers and new expression of factors and signaling molecules most commonly found in developing bone. We and others have established a critical role for matrix metalloproteinases (MMPs) in the progression of arterial calcification. MMPs are highly expressed in calcifying arteries, and we have demonstrated that reducing their activity with broad spectrum and synthetic inhibitors can prevent experimental calcification both in vitro and in vivo. In preliminary data for the current proposal, we show that MMP-3 (stromelysin-1) is the most strongly induced of all metalloproteinases in calcifying rodent aortas. Reducing MMP-3 activity decreases calcium accumulation in cultured human and rodent SMCs and in a rat aorta organ culture model. Aortas from MMP-3-deficient mice are protected from medial calcification in organ culture and in vivo. Additionally, we have recently shown that MMP-3 can decrease levels of ENPP1 that generates anti- calcifying pyrophosphate (PPi) from ATP. The location of MMP-3 activity required for calcification, whether local or systemic, and its contribution to SMC transformation is not currently known and this may act as a barrier to development of appropriate inhibitors for use in clinical trials. Based on our data and those of others, we propose the overarching hypothesis that MMP-3 contributes to medial artery calcification by promoting transformation of vascular SMCs and altering the balance of circulating stimulators and inhibitors. Reducing MMP-3 activity will slow the progression of arterial calcification in patients. As we test this hypothesis we will 1) delineate the contribution of MMP-3 to phosphate-induced SMC transformation and determine whether it can promote calcification by degrading ENPP1. 2). Determine whether MMP-3 promotes in vivo arterial calcification through local actions on medial SMCs or by altering the balance of circulating stimulators and inhibitors, and 3) Determine whether inhibiting MMP activity with doxycycline can prevent progression of peripheral artery calcification in a clinical trial. As we progress through these aims we will answer critical questions about the role of MMPs in arterial calcification and the potential for a dedicated clinical trial to assess the MMP inhibition strategies aimed at reducing calcification and improving outcomes in our patients with arterial disease.